Vascular permeability factor (hereinafter "VPF") is a 34-43 kilodalton glycoprotein and is a potent inducer of microvascular hyperpermeability. As such, VPF is believed to be responsible for the vascular hyperpermeability and consequent plasma protein-rich fluid accumulation that occurs in-vivo with solid tumors and ascites tumors [Senger et al., Science 219: 983-985 (1983); Dvorak et al., J. Immunol. 122: 166 (1979); Nagy et al., Biochim. Biophys. Acta. 948: 305 (1988); Senger et al., Federation Proceedings 46: 2102 (1987)]. On a molar basis, VPF increases microvascular permeability with a potency which is typically 50,000 times that of histamine [Senger et al., Cancer Res. 50: 1774-1778 (1990)].
VPF is also known as vascular endothelial growth factor (or "VEGF") because of its mitogenic effects on vascular endothelial cells (hereinafter "EC"). VPF is a specific EC mitogen and stimulates endothelial cell growth and angiogenesis [Conn et al., Proc. Natl. Acad. Sci. USA 87: 2628-2632 (1990); Ferrara et al., Biochem. Biophys. Res. Comm. 161: 851-858 (1989); Gospodarowicz et al., Proc. Natl. Acad. Sci. USA 86: 7311-7315 (1989); Keck et al., Science 246: 1309 (1989); Leung et al., Science 246: 1306 (1989); Connolly et al., J. Clin. Invest. 84: 1470-1478 (1989)]. In addition, VPF also exerts a number of other effects on endothelial cells in-vivo. These include: an increase in intracellular calcium; a stimulation of inositol triphosphate formation; a provocation of von Willebrand factor release; and a stimulation of tissue factor expression [Brock et al., Am. J. Pathol. 138: 213 (1991); Clauss et al., J. Exp. Med. 172: 1535 (1990)].
VPF is typically synthesized and secreted in-vivo by a variety of cultured tumor cells, transplantable animal tumors, and many different primary and metastatic human tumors [Dvorak et al., J. Exp. Med. 174: 1275-1278 (1991); Senger et al., Cancer Res. 46: 5629-5632 (1986); Plate et al., Nature 359: 845-848 (1992); Brown et al., Am. J. Pathol. 143: 1255-1262 (1993)]. Solid tumors must generate a vascular stroma in order to grow beyond a minimal size [Folkman, J. and Y. Shing, J. Biol. Chem. 267: 10931-10934 (1992)]. In addition, it has been found that some normal embryonic and normal adult tissues express mRNA for VPF synthesis [Phillips et al., Endocrinology 127: 965-967 (1991); Berse et al., Mol. Biol. Cell. 3: 211-220 (1992); Breier et al., Development 114: 521-532 (1992)]. VPF also has been reported to induce monocyte procoagulant activity and cell migration [Clauss et al., J. EX. Med. 172: 1535-1545 (1990)]. For these reasons, VPF today is believed able to induce angiogenesis generally and tumor stroma formation particularly in two ways: directly, as an endothelial cell growth factor; and, indirectly, by causing leakage of plasma proteins including fibrinogen (which clots to form an extravascular fibrin gel matrix and stimulates neovascularization with the subsequent deposition of mature connective tissue stroma). Consistent with this mechanism of action, monoclonal antibody against VPF has been shown to suppress growth and decrease the density of blood vessels in experimental tumors [Kim et al., Nature 362: 841-844 (1993)].
Structurally and chemically, VPF is a dimeric protein which is produced in-vivo in at least four major variant forms as a result of alternative splicing of mRNA [Houck et al., Mol. Endocrinol. 5: 1806-1814 (1991); Keck et al., Science 246: 1309-1312 (1989); Leung et al., Science 246: 1306-1309 (1989); Tischer et al., Biochem. Biophys. Res. Commun. 165: 1198-1206 (1989)]. In addition, complimentary DNAs coding for VPF obtained from human, bovine, rat, and guinea pig sources have been cloned [Berse et al., Mol. Biol. Cell. 3: 211-220 (1992); Conn et al., Proc. Natl. Acad. Sci. USA 87: 2628-2632 (1990)].
Under in-vivo conditions, the events induced by VPF apparently result from a direct interaction between the intact VPF molecule and specific endothelial cell surface receptors, two of which (flt-1 and kdr) have recently been identified [deVries et al., Science 255: 989-991 (1992); Terman et al., Biochem. Biophys. Res. Commun. 187: 1579-1586 (1992)]. Investigation of VPF and its receptors in tumors and vasculature remains an area of primary interest [Brown et al., Cancer Res. 53: 4727-4735 (1993); Brown et al., Am. J. Pathol. 143: 1255-1262 (1993)].
It will be noted and appreciated also that many research investigations reported in the scientific literature have employed antibodies raised against VPF in order to identify and characterize the functions, properties, and attributes of the VPF molecule in-vivo. Merely illustrating the range and variety of these investigations and published reports are the following: Preparation of antibodies [U.S. Pat. No. 5,036,003]; monoclonal antibodies to suppress growth and decrease density of blood vessels in tumors [Kim et al., Nature 362: 841-844 (1993)]; inhibition of tumor growth and metastasis by antibody to VPF [Asano et al., Cancer Res. 55: 5296-5301 (1995)]; inhibition of VPF activity with specific antibodies [Sioussat et al., Arch. Biochem. Biophys. 301: 15-20 (1993)]; the structure of solid tumors and their vasculature [Dvorak et al., Cancer Cells 3: 77-85 (1991)]; and the distribution of VPF in tumors and the concentration of VPF in tumor blood vessels (Dvorak et al., J. Exp. Med. 174: 1275-1278 (1991)]. The text of each of these publications is expressly incorporated by reference herein.
Despite the ever-increasing body of knowledge regarding VPF, its functions, and its role in tumor angiogenesis, no meaningful or substantial information regarding the use and preparation of specific anti-VPF antibodies as markers of bound VPF in-vivo existed prior to 1991; and no diagnostic or therapeutic molecule specific for VPF bound in-vivo to tumor associated blood vessels existed prior to applicants' parent invention comprising antibody-effector conjugated compounds targeting bound VPF which is the subject matter of U.S. patent application Ser. No. 779,384 filed Oct. 18, 1991 as well as the presently continuing applications U.S. Ser. No. 327,709 filed Oct. 24, 1994 and U.S. Ser. No. 464,956 filed Jun. 5, 1996. Thus, the development of additional improvements comprising anti-VPF conjugated molecules prepared as an admixture of different types and having the specific capability to bind to one or more spatially exposed regions of VPF bound in-vivo will be recognized as a major advance and unexpected improvement in this field.